I. Field of the Invention
This invention relates to a culture medium for propagating various mammalian cell lines. In particular, it is directed to a chemically defined culture medium containing elevated levels of certain amino acids. The medium supports the growth of mammalian cells at high densities as monolayers or in suspension, and the medium may be used for small-scale or for large-scale propagation of mammalian cells.
II. Description of Background and Related Art
A wide variety of cell types have been isolated from mammalian tissues and organs. Most of these cells can be propagated in a liquid medium that contains all of the nutrients and growth factors required to support the growth of the cells. Different cell types often have varied nutritional and growth factor requirements, and thus many distinct media have been developed to meet the differing needs of each cell type. Some of the original media developed for culturing mammalian cells require the addition of serum extracted from blood. Sera commonly used as additives include FBS (fetal bovine serum) and FCS (fetal calf serum). The use of this serum has been problematic primarily because of its prohibitive cost. It is usually the most expensive ingredient in the culture medium. In addition, serum frequently contains a large number of uncharacterized ingredients, and the quality and quantity of these ingredients can vary between lots and between vendors. Thus, serum may introduce particular components into the culture medium that may inhibit or otherwise affect the growth of cells that are cultured in the medium. To overcome these problems, there has been a trend towards developing serum-free media for propagating mammalian cell cultures. One common method for achieving this goal has been to use publicly available media that historically have been supplemented with serum, and to supplement instead with various components including vitamins, minerals, trace elements, hormones, serum albumin, lipids/fatty acids, and/or hormones and other growth factors. A review of many of the media developed for culturing eukaryotic cells is presented in Ham and McKeehan (Meth. Enz., 58:44 [1979]; see also Bottenstein et al., Meth. Enz., 58:94 [1979]).
Several cell lines originating from Chinese hamster have been used successfully to produce recombinant proteins. A variety of cell culture media have been specifically developed for culturing one or more of these cell lines.
Hamilton and Ham (In Vitro, 13:537 [1977]) developed a protein-free synthetic medium for clonal growth of Chinese hamster ovary cell lines. This medium is based on commercially available F12 medium (Sigma Chemical Company, St. Louis, MO) but contains increased amounts of calcium chloride and glutamine, is supplemented with 19 organic ions and selenium, and has a decreased level of cysteine.
Carver et al. (In Vitro, 19:699 [1983]) prepared several Chinese hamster ovary cell line culture media. The media are based on either F12 or Eagle's minimum essential medium (MEM), and are modified by the addition of various supplements, and/or altering the concentration of serum added to each medium.
Gasser et al. (In Vitro Cell. Devel. Biol., 88 21:588 [1985]) developed a serum-free medium for culturing Chinese hamster ovary cell lines. The medium is a 1:1 mixture of F12:MEM supplemented with transferrin, insulin, and selenium.
Mendiaz et al. (In vitro Cell. Devel. Biol., 22:66 [1986]) disclosed a defined medium for culturing CHO cells. The medium is FI2 medium supplemented with various components including seven amino acids.
While some of the above cited work has resulted in production of cell culture media for Chinese hamster cell lines that are chemically defined and serum-free, the media may be expensive and/or laborious to prepare. In addition, these media have not been optimized for high density growth of cell lines that have been genetically engineered to produce and secrete proteins and other biological compounds of commercial interest. Thus, there is a continuing need in the art for developing cell culture media that are simple to prepare, economical, and that provide all of the necessary nutrients and growth factors, at suitable concentrations, to optimize the growth of the cells.
Accordingly, it is an object of this invention to prepare culture media that support the growth of various mammalian cell lines. Another object of this invention is to provide culture media that are preferably serum-free and that may be used in either small-scale cultures or preferably in large-scale commercial production of mammalian cells. A further object of this invention is to provide media that are suitable for high density culturing of mammalian cells. Yet another object of this invention is to provide a cell culture medium that serves to increase the yield of biological products produced by the cells cultured in the media. A further object of this invention is to provide culture media that are completely chemically defined. Another object of this invention is to provide culture media that are economical to produce. One other object of this invention is to provide a method of culturing mammalian cells in a suitable medium for growth and production of biological compounds of interest.
These and other objects will be apparent to one of ordinary skill in the art.